Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering
Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering
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DOI：
10.1016/j.ijbiomac.2016.03.003
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发表时间：
2016-06-01
影响因子：
8.2
通讯作者：
Ward, Richard J.
中科院分区：
文献类型：
--
作者：
Alponti, Juliana Sanchez;Maldonado, Raquel Fonseca;Ward, Richard J.
为了提高枯草芽孢杆菌中温木聚糖酶A(XynA)的热稳定性，利用5个单突变体(522E、S27E、N32D、N54E和N181R)构建了一个随机组合文库，并对该文库进行了耐热突变筛选，确定了一个双突变(S22E/N32D)。6个突变体均在大肠杆菌(BL21)中表达并纯化。木聚糖酶活性测定表明，所有突变株的最适催化温度(T-opt)均为55℃，且除S27E突变株外，其比活力均高于野生型XynA。在55℃下失去50%活性的时间(t(50))按S22E/N32D>；N181R>；S22E>；野生型>；S27E=N32D，-IN54E的顺序减少。根据圆二色椭圆度变化监测的热变性曲线，估算了天然状态和变性状态之间的范氏变性热变(AHND)、熔融温度(TM)和恒压热容(ACP)。328K(AG328)S22E/N32D>；N181R>；S22E>；野生型>；S275E=N32D的Gibbs自由能变化的降序与N54E相近。与耐热性结果有很好的相关性，并由AHND的变化主导，这与耐热突变体中氢键的增加一致。(C)2016爱思唯尔B.V.保留所有权利。
Aiming to improve thermostability of the mesophilic xylanase A from Bacillus subtilis (XynA), five single mutants (522E, S27E, N32D, N54E and N181R) were used to construct a random combinatorial library, and screening of this library for thermostable XynA variants identified a double mutant (S22E/N32D). All 6 mutants were expressed in Escherichia coli (BL21) and purified. Xylanase activity showed all mutants have an optimum catalytic temperature (T-opt) of 55 degrees C, and with the exception of the S27E mutant, a higher specific activity than the wild-type XynA. The time for loss of 50% activity at 55 degrees C (t(50)) decreased in the order S22E/N32D >N181R > S22E > Wild-type > S27E= N32D,--IN54E. The values of the van't Hoff denaturation enthalpy change (AHND), melting temperature (Tm) and heat capacity at constant pressure (ACp) between the native and denatured states were estimated from thermal denaturation curves monitored by circular dichroism ellipticity changes. The decreasing order of Gibbs free energy change at 328 K (AG328) S22E/N32D > N181R > S22E> Wild-type >S275E=N32D approximate to N54E. correlates well with the thermotolerance results, and is dominated by changes in AHND which is consistent with increased in hydrogen bonding in the thermostable mutants. (C) 2016 Elsevier B.V. All rights reserved.